We have further characterized Zscan4 in mouse 2-cell embryos and in the subpopulation of mouse ES cells. We found that other 2-cell-specific genes are also highly expressed in Zscan4(+) cells. This indicates that the subpopulation of ES cells marked by Zscan4 expression shows some resemblance to 2-cell embryos, whereas the majority of ES cells show similarity to the Inner Cell Mass (ICM) cells in blastocysts. By carrying out cell lineage tracing experiments, we have found that, although Zscan4 is expressed only in 5% of ES cells in culture at a given time, essentially all the ES cells experience Zscan4-positive state by 9 passages. We are currently carrying out the Zscan4 knockdown and knockout experiments.